The mouse Lyt-3a gene, which encodes the Lyt-3.1 T-cell surface alloantigen of the C.AKR strain, has been cloned, and the nucleotide sequence of its exons and more than 2 kb of 5' flanking sequence have been determined. The gene extends over approximately 16 kb of DNA and consists of six exons encoding leader, leader plus V-like domain, membrane-proximal, transmembrane, and cytoplasmic domains. The only difference between the coding region of the Lyt-3a gene and the cDNA sequences reported for Lyt-3b (Nakauchi et al. 1987. Panaccio et al. 1987) is at position 77 of the mature protein where Lyt-3a encodes serine and Lyt-3b encodes arginine. This substitution must therefore be the basis for the serological distinction between the Lyt-3.1 and Lyt-3.2 alloantigens. Potential TATA and CAAT sequences, two Sp1 protein binding sites, two extended repeats of the dinucleotide, CA, a number of short inverted repeats, and an inverted segment of the mouse B1 repetitive sequence are found 5' to the Lyt-3a gene. Two consensus poly-A addition signals and a complete copy of the mouse B1 sequence are found 3' to the gene. Both B1-related regions are flanked by short direct repeats suggesting that they arose by an insertional mechanism. Cotransfection of the Lyt-3a gene together with a cloned Lyt-2a gene resulted in expression of both Lyt-2 and Lyt-3.1 on the surface of Ltk- and BW5147 cells.(ABSTRACT TRUNCATED AT 250 WORDS)